(1) Technical Field
The present invention relates generally to the field of cellular monitoring and more specifically to methods for assessing cellular metastatic potential through impedance monitoring.
(2) Related Art
The growth of large solid tumors is dependent on an aberrant cell exhibiting uncontrolled proliferation and expressing the genes required to recruit endothelial cells to undergo angiogenesis, allowing the mass to grow beyond the constraints of passive diffusion (10). Notwithstanding, these behaviors alone do not result in multiple tumor formation—the hallmark of most malignant cancers. For secondary tumor formation via a blood-borne route, some tumor cells leave the primary mass, disrupt the basement membrane of capillary endothelial cells, affect the retraction of the endothelial cells, move through the intercellular junctions, and enter the circulatory system. After moving some distance from the primary tumor, these cell then attach to or become arrested near distant endothelial cells, traverse the spaces between these cells, disrupt the basement membrane structure, and migrate into the underlying tissue (4). Uncontrolled proliferation again ensues to form a new secondary tumor. As these activities are repeated, multiple tumors are established and, if unheeded, ultimately result in the death of the organism.
This complex in vivo metastatic behavior involves the regulation and expression of many genes involved in such diverse activities as endothelial cell binding (14), cell signaling resulting in endothelial retraction (11), the synthesis and secretion of proteolytic enzymes (1), and cell locomotion (3). These various activities can be isolated and individually observed in vitro. In addition, these components can be collectively studied using tissue culture methods similar to those introduced by Kramer and Nicolson (13). Here, a cell monolayer of bovine endothelial cells was first established and then exposed to cell suspensions of a variety of both tumorigenic and nontumorigenic cells.
Cell behavior was studied using scanning and transmission electron microscopy as well as phase-contrast timelapse microscopy. When endothelial cell monolayer were challenged with highly metastatic cell lines, such as the B16 melanoma cells, observations were recorded that showed the binding of the cells to the endothelial cell layer, the retraction of the endothelial cell junctions, and finally, the penetration of the cells through the endothelial monolayer.
This in vitro sequence of activities has been suggested to represent similar invasive activities that take place during the metastatic process in vivo. The assay provides striking images of the behavior of the metastatic cell but, by nature, is difficult to quantify and cannot provide information in real time regarding the dynamics of the process. Since its introduction, this type of assay has been modified and used extensively to monitor transendothelial migration of tumor cells in vitro. Although many of these approaches have involved qualitative microscopic observations, others have employed radio or fluorescent labeling of the tumor cells to yield quantitative measurements. Such labeling, however, often utilizes hazardous probes or requires the use of specialized equipment in order to produce quantitative data. A need exists, therefore, for a safe and simple quantitative method of assessing metastatic cellular behavior.
Such a quantitative method is the electric cell-substrate impedance sensing (ECIS™, Applied BioPhysics, Troy, N.Y., USA) method first described by Giaever and Keese (6) in 1984. Since then, the method has been used to provide information regarding real-time changes in cell morphology, including cell substrate interactions, cell motility, and cell layer barrier function (7-9, 12, 15, 16, 18, 19).
The purpose of the research leading to the present invention was to determine the efficacy of such an ECIS™-based assay in assessing the metastatic potential of a cell. Concomitant methods would include, for example, methods for testing the efficacy of anti-cancer therapies and methods for establishing indicators of metastatic potential.